OP0107 NEUTROPHILS DISPLAY SHARED AND TISSUE-SPECIFIC PHENOTYPES IN INFLAMMATORY ARTHRITIS AND COLITIS
Authors: G. Osmanusta, T. Exner, S. E. Leonhardt, H. M. Lorenz, G. Wabnitz, W. Merkt, A. Gauss, R. Grieshaber-Bouyer
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Neutrophils are essential in physiological and pathological immune processes through cytokine release, formation of neutrophil extracellular traps and crosstalk with the adaptive immune system. While neutrophils display varying phenotypes as a function of maturation stage, organ and inflammatory trigger, it is poorly understood which neutrophil phenotypes are organ-specific and which are shared. Spondyloarthropathies are a family of diseases with highly variable organ involvement, including colitis and arthritis. <h3>Objectives</h3> Our goal was to understand how neutrophils in the inflamed joint and intestine differ from those in blood and which neutrophil states observed in organs are overlapping and distinct. <h3>Methods</h3> We prospectively collected healthy donor blood, paired blood and synovial fluid from patients with active inflammatory arthritis in which a joint aspiration was diagnostically or therapeutically required, and colon biopsies from patients with inflammatory bowel disease (IBD) in which endoscopy was diagnostically required. Neutrophils were enriched from blood using ACK lysis and from colon biopsies by collagenase digestion. Synovial fluid was diluted, filtered and lysis was performed on samples with visible blood contamination. Technical validation of lysis and digestion of blood cells were performed to control for the influence of these procedures on marker expression. Flow cytometry was applied on the day of sample acquisition to fresh, surface-stained neutrophils to measure protein expression. A multiplex sandwich assay utilizing fluorescent antibody-conjugated beads was used to quantify inflammatory cytokine abundance in concomitant patient sera. <h3>Results</h3> In blood, neutrophils from patients with arthritis or inactive IBD did not show prominent surface protein differences compared to healthy donors. In patients with active colitis, expression of HLA-DR and CCR5 was elevated, while CXCR2 expression was reduced. Compared to blood, neutrophils from colon tissue showed downregulation of CD16, CD62L, ICAM-1, IL-17RA and CXCR2 and upregulation of the aging marker CXCR4. These changes were observed irrespective of disease activity. In addition, neutrophils from colon of patients with active inflammation, but not patients in endoscopic remission, showed a marked increase in expression of CD64 and HLA-DR, and the downregulation of CD62L and CXCR2 was more pronounced. We also identified an expanded population of CCR5+ neutrophils in colon tissue, which was more prominent in inflamed samples. While observable in both tissues, neutrophils in synovial fluid showed a stronger upregulation of HLA-DR and CXCR4 compared to colon. Upregulation of CCR5 and CD64, as well as the downregulation of CD16 were less pronounced. In contrast, synovial fluid neutrophils showed an upregulation of ICAM-1, while this marker was downregulated in the colon. Additionally, synovial fluid neutrophils showed an upregulation of TNF-RII, which was absent in blood and colon tissue. In serum, we observed greater abundance of IL-1β, IFN-α2, IL-33, sTNF-RI and sTNF-RII in endoscopically active IBD patients compared to inactive patients. In contrast, measured IL-10 levels were lower in these patients, underscoring the shift to a pro-inflammatory cytokine environment. Independent of disease activity, measured TNF-α levels were higher in patients with Crohn's disease compared to ulcerative colitis. <h3>Conclusion</h3> Tissue infiltrating neutrophils display a range of phenotypes in both inflammatory arthritis and inflammatory bowel disease. These range from mature cells lacking activation markers and resembling circulating neutrophils, to CD64+ HLA-DR+ CXCR4+ CXCR2- CD16low aged and activated cells, as well as CCR5+ pro-NETotic cells, in varying proportion. Elevated serum levels of innate- associated cytokines and an increased activation phenotype during active colitis suggest a strong neutrophil involvement in active IBD. Organ-specific differences in neutrophil effector states may require special approaches for therapeutic intervention. <h3>Acknowledgements</h3> This work was supported by funds from the state of Baden-Wuerttemberg within the Centers for Personalized Medicine Baden-Wuerttemberg (ZPM), a physician-scientist development grant from the Medical Faculty Heidelberg and a research grant from the German Society for Rheumatology (DGRh). We are thankful to the physicians and technicians from the departments of Medicine IV and V who made the coordination and execution of this project possible. <h3>Disclosure of Interests</h3> None Declared.